Characterization of hormonal influences on Chlamydia development. Naturally fluctuating levels of the hormones, estrogen and progesterone, alter the occurrence and/or severity of sexually transmitted infections (STIs). An estimated 100 million women worldwide use hormonal contraceptives (HCs) containing combinations of synthetic estrogen and/or progesterone. Several epidemiological studies correlate HC use to increased STI transmission, including Chlamydia trachomatis infections. C. trachomatis annually causes 2,800,000 infections in the US and remains the most frequent bacterial STI worldwide. The non-invasive, sexually transmitted C. trachomatis serovars D-K are obligate intracellular pathogens that replicate primarily within genital tract (GT) luminal and glandular epithelial cells. If untreated, these organisms can ascend the GT, evoking pelvic inflammatory disease, infertility and ectopic pregnancy in infected women. Clinical trials, animal models and in vitro studies all suggest that estrogen enhances chlamydial infection by multiple mechanisms. Steroid hormones have both direct and indirect effects on endometrial epithelial cells and also induce endometrial stromal cells to release effector molecules that regulate growth and maturation of uterine epithelial cells. Previously we investigated the effects of hormones on chlamydial infection in an endometrial epithelial cell (Ishikawa, IK)/stromal cell (SHT-290) co-culture system, which provides a unique opportunity to dissect complex hormone/stromal cell/epithelial cell interaction in a simplified in vitro setting. Data from these studies indicate that: i) membrane-associated estrogen receptors help mediate C. trachomatis serovar E entry into host genital epithelial cells; ii) estrogen receptor signaling facilitates intracellular development of C. trachomatis; iii) estrogen-stimulated endometrial stromal cells secrete effectors that indirectly aid intracellular chlamydial development in genital epithelial cells, and; iv) progesterone antagonizes the effects of estrogen on chlamydial development. This proposal will test three independent, but related, hypotheses. First, in estrogen-exposed co-cultures, the stromal cell effectors osteopontin, vimentin and fibronectin will modulate C. trachomatis infection; with osteopontin and/or vimentin enhancing infection, while fibronectin reduces infection. Second, synthetic sex hormone derivatives will affect chlamydial development similarly to natural estrogen and/or progesterone in IK/SHT-290 co-cultures. Third, estrogen will enhance C. muridarum infection both in IK/SHT-290 co-cultures and in ovariectomized mice compared to progesterone exposure. Data from these experiments will: i) provide further characterization of a physiologically-relevant in vitro infection model of the female endometrium; and ii) increase understanding of how hormones modulate chlamydial infection, transmission and reproductive pathology.